Crystal structure of a class III adenylyl cyclase-like ATP-binding protein from Pseudomonas aeruginosa.

In many organisms, the ubiquitous second messenger cAMP is formed by at least one member of the adenylyl cyclase (AC) Class III. These ACs feature a conserved dimeric catalytic core architecture, either through homodimerization or through pseudo-heterodimerization of a tandem of two homologous catalytic domains, C1 and C2, on a single protein chain. The symmetric core features two active sites, but in the C1-C2 tandem one site degenerated into a regulatory center. Analyzing bacterial AC sequences, we identified a Pseudomonas aeruginosa AC-like protein (PaAClp) that shows a surprising swap of the catalytic domains, resulting in an unusual C2-C1 arrangement. We cloned and recombinantly produced PaAClp. The protein bound nucleotides but showed no AC or guanylyl cyclase activity, even in presence of a variety of stimulating ligands of other ACs. Solving the crystal structure of PaAClp revealed an overall structure resembling active class III ACs but pronounced shifts of essential catalytic residues and structural elements. The structure contains a tightly bound ATP, but in a binding mode not suitable for cAMP formation or ATP hydrolysis, suggesting that PaAClp acts as an ATP-binding protein.

A Fold-Independent Interface Residue Is Crucial for Complex Formation and Allosteric Signaling in Class I Glutamine Amidotransferases.

The members of the glutamine amidotransferase (GATase) family catalyze the incorporation of ammonia within numerous metabolic pathways and can be categorized in two classes. Here, we concentrated on class I GATases, which are heteromeric enzyme complexes consisting of synthase subunits and glutaminase subunits with a catalytic Cys-His-Glu triad. Glutamine hydrolysis at the glutaminase subunit is (i) dependent on the formation of tight synthase-glutaminase complexes and (ii) allosterically coupled to the presence of the substrate at the synthase subunit. The structural basis of both complex formation and allostery is poorly understood. However, previous work on 4-amino-4-deoxychorismate synthase and imidazole glycerol phosphate synthase suggested that a conserved aspartate residue in their synthase subunits, which is located at the subunit interface close to the glutaminase catalytic triad, might be important for both features. We performed a computational screen of class I GATases from the Protein Data Bank and identified conserved and similarly located aspartate residues. We then generated alanine and glutamate mutants of these residues and characterized them by analytical gel filtration and steady-state enzyme kinetics. The results confirmed the important role of the wild-type aspartate residues for the formation of stable synthase-glutaminase complexes (in three of four cases) and the stimulation of glutaminase activity in the analyzed GATases (in all four cases). We present a model for rationalizing the dual role of the conserved aspartate residue toward a unifying regulation mechanism in the entire class I GATase family.

Rosetta:MSF: a modular framework for multi-state computational protein design.

Computational protein design (CPD) is a powerful technique to engineer existing proteins or to design novel ones that display desired properties. Rosetta is a software suite including algorithms for computational modeling and analysis of protein structures and offers many elaborate protocols created to solve highly specific tasks of protein engineering. Most of Rosetta's protocols optimize sequences based on a single conformation (i. e. design state). However, challenging CPD objectives like multi-specificity design or the concurrent consideration of positive and negative design goals demand the simultaneous assessment of multiple states. This is why we have developed the multi-state framework MSF that facilitates the implementation of Rosetta's single-state protocols in a multi-state environment and made available two frequently used protocols. Utilizing MSF, we demonstrated for one of these protocols that multi-state design yields a 15% higher performance than single-state design on a ligand-binding benchmark consisting of structural conformations. With this protocol, we designed de novo nine retro-aldolases on a conformational ensemble deduced from a (ßα)8-barrel protein. All variants displayed measurable catalytic activity, testifying to a high success rate for this concept of multi-state enzyme design.

To gel or not to gel - Tuning the sulfation pattern of carrageenans to expand their field of application.

Carrageenans represent a major cell wall component of red macro algae and, as established gelling and thickening agents, they contribute significantly to a broad variety of commercial applications in the food and cosmetic industry. As a highly sulfated class of linear polysaccharides, their functional properties are strongly related to the sulfation pattern of their carrabiose repeating units. Therefore, the biocatalytic fine-tuning of these polymers by generating tailored sulfation architectures harnessing the hydrolytic activity of sulfatases could be a powerful tool to produce novel polymer structures with tuned properties to expand applications of carrageenans beyond their current use. To contribute to this vision, we sought to identify novel carrageenan sulfatases by studying several putative carrageenolytic clusters in marine heterotrophic bacteria. This approach revealed two novel formylglycine-dependent sulfatases from Cellulophaga algicola DSM 14237 and Cellulophaga baltica DSM 24729 with promiscuous hydrolytic activity towards the sulfated galactose in the industrially established ι- and κ-carrageenan, converting them into α- and ß-carrageenan, respectively, and enabling the production of a variety of novel pure and hybrid carrageenans. The rheological analysis of these enzymatically generated structures revealed significantly altered physicochemical properties that may open the gate to a variety of novel carrageenan-based applications.

Biocatalytic Conversion of Carrageenans for the Production of 3,6-Anhydro-D-galactose.

Marine biomass stands out as a sustainable resource for generating value-added chemicals. In particular, anhydrosugars derived from carrageenans exhibit a variety of biological functions, rendering them highly promising for utilization and cascading in food, cosmetic, and biotechnological applications. However, the limitation of available sulfatases to break down the complex sulfation patterns of carrageenans poses a significant limitation for the sustainable production of valuable bioproducts from red algae. In this study, we screened several carrageenolytic polysaccharide utilization loci for novel sulfatase activities to assist the efficient conversion of a variety of sulfated galactans into the target product 3,6-anhydro-D-galactose. Inspired by the carrageenolytic pathways in marine heterotrophic bacteria, we systematically combined these novel sulfatases with other carrageenolytic enzymes, facilitating the development of the first enzymatic one-pot biotransformation of ι- and κ-carrageenan to 3,6-anhdyro-D-galactose. We further showed the applicability of this enzymatic bioconversion to a broad series of hybrid carrageenans, rendering this process a promising and sustainable approach for the production of value-added biomolecules from red-algal feedstocks.

Conformational Modulation of a Mobile Loop Controls Catalysis in the (ßα)8-Barrel Enzyme of Histidine Biosynthesis HisF.

The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site ß1α1-loop (loop1) of the (ßα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of (ßα)8-barrel enzymes and beyond.

Molecular basis for the allosteric activation mechanism of the heterodimeric imidazole glycerol phosphate synthase complex.

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism. HisFH is responsible for the HisH-catalyzed hydrolysis of glutamine to glutamate and ammonia, which is then used for a cyclase reaction by HisF. The HisFH complex is allosterically regulated but the underlying mechanism is not well understood. Here, we elucidate the molecular basis of the long range, allosteric activation of HisFH. We establish that the catalytically active HisFH conformation is only formed when the substrates of both HisH and HisF are bound. We show that in this conformation an oxyanion hole in the HisH active site is established, which rationalizes the observed 4500-fold allosteric activation compared to the inactive conformation. In solution, the inactive and active conformations are in a dynamic equilibrium and the HisFH turnover rates correlate with the population of the active conformation, which is in accordance with the ensemble model of allostery.

Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids.

Imidazole glycerol phosphate synthase (ImGPS) is an allosteric bienzyme complex in which substrate binding to the synthase subunit HisF stimulates the glutaminase subunit HisH. To control this stimulation with light, we have incorporated the photo-responsive unnatural amino acids phenylalanine-4'-azobenzene (AzoF), o-nitropiperonyl-O-tyrosine (NPY), and methyl-o-nitropiperonyllysine (mNPK) at strategic positions of HisF. The light-mediated isomerization of AzoF at position 55 (fS55AzoFE ↔ fS55AzoFZ) resulted in a reversible 10-fold regulation of HisH activity. The light-mediated decaging of NPY at position 39 (fY39NPY → fY39) and of mNPK at position 99 (fK99mNPK → fK99) led to a 4- to 6-fold increase of HisH activity. Molecular dynamics simulations explained how the unnatural amino acids interfere with the allosteric machinery of ImGPS and revealed additional aspects of HisH stimulation in wild-type ImGPS. Our findings show that unnatural amino acids can be used as a powerful tool for the spatiotemporal control of a central metabolic enzyme complex by light.